Low Testosterone Concentration Improves Colonisation and Viability in the Co‐Cultured Goat Spermatogonial Stem Cell With Sertoli Cells

ABSTRACT Spermatogonial stem cells (SSCs) maintain spermatogenesis through self‐renewal and differentiation. The proliferation of SSCs in culture systems can provide a valuable source of germ cells. Several studies have investigated new reproductive technologies, including the production of transgenic animals and recombinant proteins secreted from milk in goats. While studies in other species exist, research on goat SSC culture remains limited. We investigated the impact of different testosterone concentrations on the survival and colonisation of cocultured goat SSCs with Sertoli cells. Cells were isolated from immature goats using two‐step enzymatic digestion and enriched by differential exclusion method. DMEM/F12 culture medium containing 1% antibiotic and 5% FBS, supplemented with GDNF (20 ng/mL), EGF, bFGF and LIF (10 ng/mL), was used with different testosterone concentrations (0, 60, 120 and 240 μg/mL) and cultured for 10 days. SC subpopulations were confirmed using PGP9.5 immunocytochemistry, and the expression of germ cell markers (ID‐4, UCHL‐1, THY‐1, β1‐integrin, BCL6B, VASA, PLZF and OCT‐4) was evaluated through RT‐PCR. Alkaline phosphatase activity provided additional SSC presence. The survival rate of SSCs after isolation and the number and area of colonies on Days 4, 7 and 10 were measured using an inverted microscope. The presence of PGP 9.5 antigens and germ cell markers (ID‐4, UCHL‐1, THY‐1, β1‐integrin, BCL6B, VASA, PLZF and OCT‐4) was confirmed by immunocytochemistry and RT‐PCR, respectively. According to the results, the group with 60 μg/mL testosterone had the highest number and area of colonies. The number of colonies in the 60 μg/mL testosterone group was significantly higher than the control group ( p < 0.05), but no significant difference was observed compared to other groups ( p ≥ 0.05). This study suggests that a low testosterone concentration (60 μg/mL) is optimal for goat SSC colonisation and viability in coculture with Sertoli cells, potentially leading to advancements in goat reproductive technologies.